Fluorescent molecular probes that can label, detect, or image specific proteins serve as a powerful tool for developing in-vitro proteomic assays, for identifying disease biomarkers, as well as for tracking proteins in complex environments.
Fluorescent molecular sensors have become valuable tools in the analytical biosciences owing to their sensitive detection mode, down to the level of a single molecule, the feasibility of naked eye visualization, their versatility, and their small size, which enable them to penetrate the cell membrane and track the rise and fall of various bio-analytes within living cells. Although fluorescent sensors that utilize photo-induced electron transfer (PET), electronic energy transfer (EET) (or fluorescence resonance energy transfer (FRET), and internal charge transfer (ICT) processes have been developed and used to detect various proteins, most of them suffer from a high background signal that complicates their use in complex biochemical mixtures and within cells.
There is a growing interest in developing “genetically targeted fluorescent molecules”, namely, small molecule-based fluorescent probes that can bind to short, peptide motifs on the protein of interest and, in doing so, enable the protein's labeling or detection in complex biological environments such as within live cells. Such sensors provide an alternative to using recombinant technology to create a fusion protein comprising the protein of interest with fluorescent proteins (FPs) (e.g., green fluorescent proteins or GFPs) whose large size can interrupt the normal function of many proteins. These genetically targeted probes have already become commercial, for example, the FlAsH-and-ReAsH probes for the selective labeling of tetra-cysteine motifs that are now sold online by Life Technologies.
Genetically encoded fluorescent proteins (FPs) have revolutionized the study of biology by allowing one to track protein expression and localization in living cells at spatial and temporal resolution. This method, however, involves the use of very large protein that can interfere with the normal function of the labeled protein. Over the last few years, it has been demonstrated that this problem can be circumvented by expressing the proteins with a very short peptide sequence to which a small fluorescent molecular sensor, termed “genetically-targeted sensors” can attach. Sensors that can bind to an oligohistidine sequence (i.e. His-tag) with high affinity and can be applied for labeling and detecting a wide range of His-tagged proteins in living cells.
The histidine tag is currently the most widely used tag in protein purification. It is typically composed of six or ten histidine residues fused at the amino or carboxyl terminus of a protein. Recombinant proteins containing a histidine tag are commonly purified on a matrix with nickel(II)-nitrilotriacetate (Ni-NTA) complexes that are prepared from nickel(II)-activation of nitrilotriacetic acid (NTA). In addition to protein purification, this technology has been used in label-free surface plasmon resonance (SPR) biosensors for biomolecular interaction analysis that involves histidine-tagged proteins.
Protein surface recognition by synthetic receptors is an important research direction in the areas of bioorganic and medicinal chemistry, particularly due to the ability of such receptors to disrupt the interactions between two proteins. Such systems can be obtained, for example, by mimicking essential interacting motifs (e.g., α-helices) of one of the protein partners. Alternatively, they may be inspired by the structure of antibodies and consist of larger receptors that complement hydrogen-bonding, hydrophobic, and charged groups on the surface of the target proteins. Despite these significant advances, protein surface recognition by synthetic agents remains challenging owing to the immense complexity of the biological targets. Specifically, protein surfaces are generally large and flat, and lack well-defined grooves and pockets that can serve as templates for designing synthetic binding partners.
Similar challenges hamper the development of fluorescent molecular sensors that can track changes that occur on the surfaces of specific proteins. If available, such systems would facilitate detecting protein modifications and binding interactions, which are difficult to sense with the current luminescent probes. Chromophoric protein surface receptors, for example, which have been used to discriminate among protein surfaces, can generally do so in the form of cross-reactive sensor arrays that are inherently not specific. Fluorescent molecular sensors and probes, on the other hand, which can efficiently detect, label, and image specific proteins, are normally designed to target only small and well-defined recognition domains. Tracking protein surface modifications by such sensors is therefore indirect, namely, the modifications must induce a significant change at the probe's binding site.
Herein, a step toward circumventing the challenges associated with sensing surfaces of specific proteins with synthetic receptors is demonstrated. In particular, it is demonstrated how the attachment of protein surface receptors to genetically targeted small molecules can afford fluorescent sensors that respond to changes in the surfaces of affinity-labelled proteins, upon binding to metal ions, small molecules, and protein partners. It is herein demonstrated how combination of flexible linker with a modifiable synthetic receptor enables the design of various sensors that match different regions on the surface of various proteins. The way this method could be used to sense surface modifications of unlabeled proteins is also presented.